Field of the Invention
The present invention relates to a fast method for the detection and the quantification of a nucleic acid, DNA or RNA. Specifically, the invention provides a method for detecting and quantifying the presence of a specific nucleic acid molecule which is based on physical and electronic treatments. The method of the invention is particularly useful for applications as diverse as detection of abnormal chromosome distributions or gene expression analysis.
Description of Related Art
The detection and quantification of nucleic acid sequences is of importance for a wide range of commonly utilized biological applications. Applications include the fields of in vitro diagnostics, including clinical diagnostics, research in the fields of molecular biology, high throughput drug screening, veterinary diagnostics, agricultural-genetics testing, environmental testing, food testing, industrial process monitoring and insurance testing. In particular, demonstration of the presence of a specific DNA sequence in a physiological sample constitutes, at the present time, the major line of development of diagnostic methods, e.g. for identifying the probability of bacteria of developing antibiotic resistance, genetic abnormalities, the risks of cancer associated with genetic modifications and viral infections, such as infections associated with HIV or with hepatitis viruses (see for example Zhang et al., Nature, 358: 591-593, 1992; Turner et al., J Bacteriol, 176(12):3708-3722, 1994; Weston et al., Infection and Immunity., 77(7):2840-2848, 2009). Detection and/or quantification of a particular nucleic acid sequence is also crucial for applications as diverse as prenatal diagnostic, study of gene expression, obtention of ancient DNA, diagnoses of genetic disease, detection of sequence polymorphisms, quantification of DNA repeats, detection of pathogens, identification of genetically-modified organisms etc. (see e.g. WO 02/31463; WO 2006/058395; U.S. Pat. No. 5,705,365; U.S. Pat. No. 5,840,487; U.S. Pat. No. 5,858,658; U.S. Pat. No. 6,453,245; U.S. Pat. No. 7,919,247; Verjat et al., Biotechniques, 37(3): 476-481, 2004; Vural, Afr. J. Biotechnol., 8(20): 5163-5168, 2009; van der Meide et al., J. Clin. Microbiol., 43(11): 5560-5566; 2005; Fan et al., Proc. Natl. Acad. Sci. USA, 105(42): 16266-16271, 2008; Buehler et al., Methods, 50: S15-S18, 2010; Clark et al., Genome Res., 11: 1594-1602, 2001; Vernon et al., BMC Infect Dis, 3: 12, 2003; Piepenburg et al., PLoS Biol., 4(7): e204, 2006).
Several techniques have been developed over the years, all relying on the detection of a hybrid molecule between the target nucleic acid molecule and a specific labelled probe. Today, the most widely used methods to detect nucleic acids are based on the polymerase chain reaction (PCR). Real-time PCR for example is used to amplify and simultaneously quantify a targeted DNA molecule.
A crucial aspect in any detection and/or quantification method of nucleic acid is sensitivity. The first techniques developed required large sample sizes. This problem was alleviated by the amplification step in the PCR method. PCR-based methods thus allowed the detection of very small amounts of nucleic acids by amplifying the target nucleic acid (Hague et al., BMC Biotechnol., 3: 20, 2003). In biological research, PCR has thus accelerated the study of testing for communicable diseases. Medical applications of PCR include identifying viruses, bacteria and cancerous cells in human tissues. PCR can even be used within single cells, in a procedure called in situ (in-site) PCR, to identify specific cell types. PCR can also be applied to the amplification of RNA, a process referred to as reverse transcriptase PCR (RT-PCR). RT-PCR is similar to regular PCR, with the addition of an initial step in which DNA is synthesized from the RNA target using an enzyme called a reverse transcriptase. A wide variety of RNA molecules have been used in RT-PCR, including ribosomal RNA, messenger RNA and genomic viral RNA.
However, all the methods developed so far suffer from serious drawbacks. In particular, they all make use of labeled nucleotides (e.g. labeled with fluorescence), thus contributing to seriously increasing the overall costs. Moreover, amplification of the target sequence is time consuming, increases the probability of errors, and is highly prone to contamination.